PROJECT SUMMARY Pancreatic adenocarcinoma (PDA) is on track to be the second leading cause of cancer related deaths in the United States by 2020. Human PDA is often found at later stages of tumor formation, is refractory to many frontline therapies, and has a low (8%) 5-year survival rate. Mouse models have made research into PDA more feasible and accurate, though the models frequently fail to recapitulate the tumor heterogeneity in human PDA. Recently, Dr. Katelyn Byrne at the University of Pennsylvania generated a tumor clone library derived from the spontaneous mouse model of PDA. These clones cover the spectrum of immune cell heterogeneity seen in human PDA, making them a vital and powerful tool to better understand how these tumors form and respond to immunotherapies. Importantly, Dr. Byrne has found a dichotomy between T cell infiltrated tumor clones and T cell excluded tumor clones in their responsiveness to checkpoint blockade therapies, mimicking human PDA patient clinical responses. Immune checkpoint blockades target negative regulatory immune pathways constraining effector function, reinvigorating antitumor immunological functions. Surprisingly, many mechanistic aspects of checkpoint blockade remain poorly characterized, likely due to the variety of immune cell populations and anatomical locations at which checkpoint blockade therapies can act. I believe our lab will be uniquely situated to address questions concerning the spatial/anatomical localizations of immune cells during checkpoint blockade, since the Germain lab specializes in cutting-edge high resolution microscopic techniques. I have established a collaboration with Dr. Byrne to use her tumor clones to interrogate the mechanisms behind checkpoint blockade therapies, especially in relation to treatment-refractory tumors. I hypothesize that a spatio-temporal analysis of infiltrating immune cell subsets in the tumor microenvironment and tumor draining lymph node during checkpoint blockade will result in mechanistic insight into the dynamic reprogramming and localization of these cells, which could reveal novel targets for rational immunotherapeutic approaches. In Aim 1, I will determine the functional anatomic site(s) of checkpoint blockade therapy in relation to T cell proliferation, priming, and localization. In Aim 2, I will elucidate the mechanisms by which myeloid cells influence T cell inclusion/exclusion phenotypes in the tumor microenvironment. In Aim 3, I will evaluate the generalizability of my key findings from Aims 1 and 2 in additional cancer models, with the hope of discovering fundamental truths that are broadly applicable. Together, these studies will provide mechanistic insight into the factors driving immunosuppressive tumor microenvironments, the factors that influence therapeutic responsiveness, and has the potential for translational applications. The activities planned under this award will provide me with an essential training opportunity that will foster my growth as an independent researcher and position me for success at a government or academic institution with my own lab.